We previously identified an 11-base DNA sequence, 5'-G-G-T-A-C-C-T-A-A-C-C-3', which is important in the control of SV40 late RNA expression in vitro and in vivo. Subsequently, a series of mutants with deletions extending from SV40 map position O to 300 was prepared by nuclease BAL 31 treatment. Our transcription studies demonstrated that, in addition to the promoter domain near map position 300, there are essential DNA sequences between nucleotide positions 74 and 95 that are required for efficient expression of late simian virus 40 (SV40) RNA. Included in this SV40 DNA sequence were two of the six GGGCGG hexamers from the SV40 21 bp repeat sequences and an 11-nucleotide segment which showed strong homology with the upstream sequences required for the efficient in vitro and in vivo expression of the histone H2A gene. This upstream promoter sequence supports transcription with the same efficiency when it is moved 72 nucleotides closer to the major late cap site. Using in vitro competition studies, we demonstrated that promoter-containing DNA fragments, which harbor neither the SV40 early or late -25 transcriptional control signals (TATA box) or the major RNA initiation sites, effectively compete for essential transcriptional factors required for SV40 transcription. The ability to compete for transcriptional factors is dependent upon sequences located within the 21 bp repeats. In addition, using an SV40-adenovirus 2 recombinant DNA, we demonstrate that the SV40 21 bp repeat region can stimulate in vitro transcription from the heterologous adenovirus 2 major late promoter (Ad2 MLP). In the absence of contigous SV40 transcription control sequences, the 21 bp repeats are capable of initiating transcription, in a bidirectional manner, from proximally located sequences.